Study of host regulation by insect viruses

众所周知，感染昆虫的杆状病毒可以有效和成功地控制宿主，但其机制仍未完全了解。本研究旨在了解杆状病毒调控宿主的机制。本研究对家蚕核多角体病毒（BmNPV）的三个基因进行了鉴定。首先，我们分析了BmNPV IE2的定位，该基因已被证明含有泛素连接酶E3活性。随着感染的进展，BmNPV IE2的产生减少，IE2形成的核灶消失。使用蛋白酶体抑制剂和E3活性缺陷突变体，我们发现IE2被泛素化，然后通过蛋白酶体途径降解。我们的研究结果表明，BmNPV IE2的E3活性可能在感染期间调节IE2的蛋白水平和细胞定位。其次，我们检查了BmNPV ORF8蛋白的定位，因为有报道称它定位于发生BmNPV复制的特定核位点。使用gfp融合的orf8，我们发现IE1和hr有助于BmNPV orf8定位到特定的核位点。最后，我们发现BmNPV - BRO蛋白通过抑制剂在细胞核和细胞质之间穿梭。BRO蛋白富含亮氨酸区域的突变导致瞬时表达蛋白的核积累，表明该区域是crm1依赖的核输出信号（NES）。相反，具有改变的NES的突变brod在感染细胞中没有显示出核积累，尽管蛋白质产量似乎减少了。RT-PCR分析表明，较低水平的蛋白质产量是由于RNA合成减少。总之，我们的结果强烈表明这些基因在BmNPV复制中起重要作用。为了更全面地了解分子机制，目前正在进行进一步的研究，包括寻找相互作用的宿主因子。

Baculoviruses that infect insects are known to control the host for their efficient and successful infection, but the mechanisms are still not fully understood. This study aims to understand the mechanism of host regulation by baculoviruses. In this study, we characterized three genes of Bambyx mori nucleopolyhedrovirus (BmNPV). First, we analyzed the localization of BmNPV IE2 that has been shown to contain ubiquitin ligase E3 activity. The production of BmNPV IE2 decreases and the nuclear foci formed by IE2 disappear as the infection progresses. Using a proteasome inhibitor and a defective mutant in E3 activity, we showed that IE2 was ubiquitinated and then degraded by the proteasome pathway. Our results suggest that the E3 activity of BmNPV IE2 might regulate the protein level and cellular localization of IE2 during infection. Secondly, we examined the localization of BmNPV ORF8 protein since it has been reported to localize to specific nuclear sites where BmNPV replication occurs. Using gfp-fused orf8, we showed that IE1 and hr facilitate the localization of BmNPV ORF8 to specific nuclear sites. Finally, we revealed that BmNPV BRO proteins shuttle between the nucleus and cytoplasm using inhibitors. Mutations on the leucine-rich region of BRO proteins resulted in nuclear accumulation of transiently expressed proteins, suggesting that this region functions as a CRM1-dependent nuclear export signal (NES). On the contrary, mutant BRO-D with an altered NES did not show nuclear accumulation in infected cells, although protein production seemed to be reduced. RT-PCR analysis showed that the lower level of protein production was due to a reduction in RNA synthesis. Together, our results strongly suggest that these genes play important roles in BmNPV replication. To more completely understand the molecular mechanisms, further investigations including search for interacting host factors are currently underway.